Among the most devastating disorders of aging are the dementing diseases. Prion diseases are age dependent, neurodegenerative illnesses that generally present as rapidly progressive dementias. Much evidence argues that prion diseases are disorders of protein conformation. The emphasis of this proposal is to investigate the molecular mechanisms that feature not only in the replication of prions but also in the pathogenesis of the disease process. Within this context, we propose to exploit our ability to synthesize both mammalian and fungal prions from proteins expressed in E. coli. We plan to produce a large number of synthetic mammalian Prions (SMP) by polymerizing N-terminally truncated PrPs comprising approximately 140 residues into amyloid fibrils. Variations in the polymerization process are likely to produce different SMP strains. These synthetic strains of infectious prions, composed of mouse, hamster, human and bovine PrPs, will be inoculated into Tg mice expressing the corresponding PrPs. The SMP strains produced in these Tg mice will be studied by mutagenic, biophysical, neuropathological and immunological approaches. Electron crystallography, fiber diffraction and molecular modeling will be used to investigate the tertiary structure of PrPSc from several different SMP strains. Such studies suggest that the infectious monomer of the RML strain of mouse prions is a trimeric complex of PrPSc molecules. The three PrPSc molecules are arranged with their b-helices facing inward and the alpha helical regions facing out toward the aqueous environment. Synthetic prions will also be produced from N-terminal prion domain of the Sup35 protein in yeast. The structures of different strains of these yeast prions will be correlated with their biological properties to learn how biological information is enciphered in the conformation of a protein. Additional yeast prions will be investigated in comparative studies. The molecular events that feature in the pathogenesis of prion disease will be investigated for several different SMP strains. Recent work suggests that prion-induced neurodegeneration proceeds through the Notch pathway. We shall determine whether a single pathway mediates the CNS degeneration observed for different SMP strains or several pathways are involved, each of which is strainspecific. The diverse skills, talents and backgrounds of the investigators in the proposed program offer an unusual opportunity to define the molecular mechanisms responsible for neurodegeneration and as such provide new approaches to investigating the more prevalent neurodegenerative disorders afflicting older people, including Alzheimer's and Parkinson's diseases.